US6562568B1 - Method, kit and apparatus comprising magnetic glass particles for the isolation of biomolecules - Google Patents
Method, kit and apparatus comprising magnetic glass particles for the isolation of biomolecules Download PDFInfo
- Publication number
- US6562568B1 US6562568B1 US09/509,750 US50975000A US6562568B1 US 6562568 B1 US6562568 B1 US 6562568B1 US 50975000 A US50975000 A US 50975000A US 6562568 B1 US6562568 B1 US 6562568B1
- Authority
- US
- United States
- Prior art keywords
- glass particles
- magnetic glass
- sample
- analyte
- reaction vessel
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 238000000034 method Methods 0.000 title claims abstract description 73
- 239000011521 glass Substances 0.000 title claims abstract description 43
- 239000002245 particle Substances 0.000 title claims abstract description 39
- 230000005291 magnetic effect Effects 0.000 title claims abstract description 28
- 238000002955 isolation Methods 0.000 title claims abstract description 15
- 239000000523 sample Substances 0.000 claims abstract description 91
- 239000012491 analyte Substances 0.000 claims abstract description 39
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 22
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 22
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 22
- 239000012472 biological sample Substances 0.000 claims abstract description 15
- 239000000725 suspension Substances 0.000 claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims description 49
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 25
- 238000002156 mixing Methods 0.000 claims description 23
- 238000010828 elution Methods 0.000 claims description 18
- 229910011255 B2O3 Inorganic materials 0.000 claims description 12
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 12
- 229910052681 coesite Inorganic materials 0.000 claims description 12
- 229910052906 cristobalite Inorganic materials 0.000 claims description 12
- JKWMSGQKBLHBQQ-UHFFFAOYSA-N diboron trioxide Chemical compound O=BOB=O JKWMSGQKBLHBQQ-UHFFFAOYSA-N 0.000 claims description 12
- 239000000377 silicon dioxide Substances 0.000 claims description 12
- 229910052682 stishovite Inorganic materials 0.000 claims description 12
- 229910052905 tridymite Inorganic materials 0.000 claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
- 230000002934 lysing effect Effects 0.000 claims description 11
- 239000011534 wash buffer Substances 0.000 claims description 11
- 108010067770 Endopeptidase K Proteins 0.000 claims description 8
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- 238000003199 nucleic acid amplification method Methods 0.000 claims description 8
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- 239000000872 buffer Substances 0.000 claims description 7
- ODINCKMPIJJUCX-UHFFFAOYSA-N calcium oxide Inorganic materials [Ca]=O ODINCKMPIJJUCX-UHFFFAOYSA-N 0.000 claims description 7
- 239000012149 elution buffer Substances 0.000 claims description 7
- KKCBUQHMOMHUOY-UHFFFAOYSA-N Na2O Inorganic materials [O-2].[Na+].[Na+] KKCBUQHMOMHUOY-UHFFFAOYSA-N 0.000 claims description 6
- 239000004365 Protease Substances 0.000 claims description 6
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 6
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 6
- 229910052593 corundum Inorganic materials 0.000 claims description 6
- 235000019419 proteases Nutrition 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 229910001845 yogo sapphire Inorganic materials 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical class NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 claims description 4
- 239000013578 denaturing buffer Substances 0.000 claims description 4
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical group [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- -1 dAl2O3 Chemical compound 0.000 claims description 2
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 claims description 2
- 239000003960 organic solvent Substances 0.000 claims description 2
- NOTVAPJNGZMVSD-UHFFFAOYSA-N potassium monoxide Inorganic materials [K]O[K] NOTVAPJNGZMVSD-UHFFFAOYSA-N 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 abstract description 42
- 238000001514 detection method Methods 0.000 abstract description 10
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 238000001179 sorption measurement Methods 0.000 description 34
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- 238000010438 heat treatment Methods 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 10
- 239000012071 phase Substances 0.000 description 10
- 239000006249 magnetic particle Substances 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- 241000606161 Chlamydia Species 0.000 description 7
- 230000009089 cytolysis Effects 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
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- 238000001816 cooling Methods 0.000 description 5
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- 239000006148 magnetic separator Substances 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
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- 239000012148 binding buffer Substances 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000003196 chaotropic effect Effects 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000007885 magnetic separation Methods 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 101100203596 Caenorhabditis elegans sol-1 gene Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 229910000272 alkali metal oxide Inorganic materials 0.000 description 2
- 229910000287 alkaline earth metal oxide Inorganic materials 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
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- 229910044991 metal oxide Inorganic materials 0.000 description 2
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- 102000004169 proteins and genes Human genes 0.000 description 2
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- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 1
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- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 108010059712 Pronase Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 101150058348 SWE1 gene Proteins 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
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- 238000003776 cleavage reaction Methods 0.000 description 1
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- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000005499 meniscus Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000002923 metal particle Substances 0.000 description 1
- 239000010445 mica Substances 0.000 description 1
- 229910052618 mica group Inorganic materials 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Inorganic materials [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 239000013615 primer Substances 0.000 description 1
- 239000002987 primer (paints) Substances 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229910052814 silicon oxide Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 150000003672 ureas Chemical class 0.000 description 1
Images
Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
- B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
- B03C1/00—Magnetic separation
- B03C1/005—Pretreatment specially adapted for magnetic separation
- B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
-
- C—CHEMISTRY; METALLURGY
- C03—GLASS; MINERAL OR SLAG WOOL
- C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
- C03C14/00—Glass compositions containing a non-glass component, e.g. compositions containing fibres, filaments, whiskers, platelets, or the like, dispersed in a glass matrix
- C03C14/004—Glass compositions containing a non-glass component, e.g. compositions containing fibres, filaments, whiskers, platelets, or the like, dispersed in a glass matrix the non-glass component being in the form of particles or flakes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
- H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
- H01F1/01—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials
- H01F1/03—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity
- H01F1/032—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of hard-magnetic materials
- H01F1/10—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of hard-magnetic materials non-metallic substances, e.g. ferrites, e.g. [(Ba,Sr)O(Fe2O3)6] ferrites with hexagonal structure
- H01F1/11—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of hard-magnetic materials non-metallic substances, e.g. ferrites, e.g. [(Ba,Sr)O(Fe2O3)6] ferrites with hexagonal structure in the form of particles
- H01F1/112—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of hard-magnetic materials non-metallic substances, e.g. ferrites, e.g. [(Ba,Sr)O(Fe2O3)6] ferrites with hexagonal structure in the form of particles with a skin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/0099—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor comprising robots or similar manipulators
Definitions
- the invention concerns a process for preparing biological samples for the subsequent detection of an analyte, in particular a nucleic acid, in this sample.
- analyte in particular a nucleic acid
- reagent kits and new devices for sample preparation and new magnetic pigments are provided.
- the sample preparation often has to meet special requirements in a method for the detection of an analyte in a biological sample.
- the analyte is often present at a very low concentration and, on the other hand, there are often many other substances in the sample which can interfere with the isolation or determination of the analyte.
- WO 96/41811 discloses a process for the isolation of an analyte, especially a nucleic acid, from a biological sample wherein the sample which contains the analyte in a liquid is contacted with magnetic particles that have an outer glass surface which is essentially free of pores or has pores with a diameter of ⁇ 10 nm, under conditions such that the analyte binds to the particle surface and the bound analyte is separated from the sample liquid.
- the process described in WO 96/46811 is very well suited to the purification of an analyte from a biological sample. However, it cannot be easily applied to an automated sample preparation.
- the object of the present invention was to provide a new sample preparation process in which the disadvantages of the state of the art are at least partially eliminated.
- a further aspect of the present invention is a process for the isolation of an analyte from a biological sample comprising the steps:
- the process according to the invention is based on the selective binding of analytes to a solid adsorption matrix in the presence of a sample lysing buffer in which the analyte that is preferably a nucleic acid such as DNA e.g. chromosomal DNA, fragmented chromosomal DNA, plasmid DNA, viral DNA etc. or RNA e.g. mRNA, tRNA, rRNA or viral RNA etc., is separated from impurities of the sample such as proteins or cell debris.
- the sample can be any biological sample e.g. a body fluid such as blood, plasma, urine etc., a tissue sample, a sample of cultured cells or such like.
- the adsorption matrix used in the process according to the invention is able to ensure the substantially selective binding of the analyte under the reaction conditions.
- a particulate adsorption matrix is preferably used which preferably contains a glass surface.
- Magnetic glass particles are particularly preferred, especially the magnetic particles described in WO 96/41811 with an external glass surface which is essentially free of pores or has pores with a diameter of less than 10 nm.
- Ferromagnetic particles are particularly preferred which have a particle size between 10 and 60 ⁇ m.
- Such particles can for example contain a core made of mica and magnetic particles immobilized thereon which is enclosed by a layer of glass. Whereas in WO 96/41811 the magnetic particles are placed in the individual reaction vessels in a solid form e.g.
- the magnetic particles are preferably used according to the invention in the form of a suspension.
- Alcoholic suspensions having a concentration of about 5 to 20 mg/ml have proven to be particularly suitable. It was surprisingly found that, despite the high specific density of the magnetic glass particles, the suspension can be very reproducibly drawn out of a storage container which enables the process to be automated.
- glass particles described in WO 96/41811 give good results in the process according to the invention, particularly good results are obtained with glass particles whose glass phase contains the following metal oxides: SiO 2 , B 2 O 3 , alkali metal oxide e.g. K 2 O or/and Na 2 O and optionally Al 2 O 3 and an alkaline earth metal oxide e.g. CaO.
- metal oxides SiO 2 , B 2 O 3 , alkali metal oxide e.g. K 2 O or/and Na 2 O and optionally Al 2 O 3 and an alkaline earth metal oxide e.g. CaO.
- the contents of these metal oxides are preferably as follows: 50 to 95 mol-% SiO 2 , 0.2 to 30 mol-% B 2 O 3 , 0 to 10 mol-% Al 2 O 3 , 0 to 20 mol-% alkaline earth metal oxide and 0.2 to 20 mol-% alkali metal oxide where the percentages are each based on the total weight of the glass phase.
- a glass phase which contains SiO 2 , B 2 O 3 , K 2 O, Al 2 O 3 and CaO has proven to be particularly suitable for the isolation of RNA.
- a glass phase which contains SiO 2 , B 2 O 3 and Na 2 O has proven to be particularly suitable for the isolation of DNA.
- the adsorption matrix is preferably added in an amount which corresponds to the minimum amount required to quantitatively bind the analyte present in the sample, in particular a nucleic acid, or the amount is somewhat larger, preferably at most 50% and particularly preferably at most 20% above this amount.
- the expected amount of nucleic acid in various type of samples can—if it is not already known—be determined in advance by common techniques e.g. phenol/chloroform extraction and subsequent measurement of the optical density.
- Step (a) of the process according to the invention comprises lysing the sample in a reaction vessel.
- This lysis is usually carried out by lysing the cells present in the sample under denaturing conditions e.g. by adding a protease and a denaturing buffer.
- Proteinase K, pronase, elastase or/and lysozyme are preferably used as the proteinase.
- the use of proteinase K is particularly preferred.
- the protease digestion is carried out in a denaturing buffer which contains a chaotropic compound e.g. urea or urea derivatives, preferably a chaotropic salt, particularly preferably a guanidinium salt such as guanidinium hydrochloride (especially for the isolation of DNA) or guanidinium thiocyanate (especially for the isolation of RNA) or a perchlorate or iodide. Concentrations in the range of 1 to 3 mol/l are preferred for guanidinium salts.
- a chaotropic compound e.g. urea or urea derivatives
- a chaotropic salt particularly preferably a guanidinium salt such as guanidinium hydrochloride (especially for the isolation of DNA) or guanidinium thiocyanate (especially for the isolation of RNA) or a perchlorate or iodide.
- the solid adsorption matrix is only added after lysing the sample. This procedure results in a significantly lower unspecific binding of undesired sample components, e.g. proteins, to the adsorption matrix.
- step (c) the analyte is selectively bound to the adsorption matrix by incubation in the lysing buffer preferably under chaotropic conditions.
- Step (d) of the process according to the invention comprises the separation of non-bound sample components from the adsorption matrix.
- the non-bound sample components are preferably removed from the reaction vessel. This can be achieved by adding and removing a wash buffer, optionally several times, which preferably contains a quantity of at least 50% (v/v) and particularly preferably of at least 60% (v/v) of a solvent that is miscible with water such as ethanol, propanol and acetone.
- Steps (c), (d) or/and (e) of the process according to the invention are preferably carried out while mixing continuously or at intervals (i.e. mixing phases alternate with phases in which the reaction vessel is at rest) without adding external means.
- This mixing is preferably carried out by rotating the reaction vessel around its longitudinal axis while reversing the direction of rotation several times.
- the mixing vessel is particularly preferably rotated exactly around its longitudinal axis and the change in the direction of rotation is carried out such that the meniscus deflection of the liquid remains below a predetermined cut-off value.
- Such mixing processes are described in WO 91/15768 and EP-A-0 435 481.
- the duration of steps (c) or/and (e) is preferably 20 min at most and comprises a continuous mixing or an interval mixing in short cycles, preferably in short cycles of preferably two minutes maximum. Particularly good results were obtained by interval mixing in a one minute cycle comprising 20 sec mixing and 40 sec resting.
- Step (e) of the process according to the invention comprises the elution of the analyte from the adsorption matrix.
- a low salt buffer that is essentially free of organic solvents can be used for this as is known from the prior art.
- the elution buffer can contain additional reagents such as enzymes e.g. enzymes used to manipulate nucleic acids such as RNases, DNases, restriction endonucleases, ligases, terminal transferases or/and polymerases. If the analyte is for example a DNA it is possible to add a DNase-free RNase during the elution in order to reduce the content of undesired RNA.
- analyte is RNA
- RNase-free DNase it is possible to add an RNase-free DNase during the elution.
- Other enzymes such as restriction endonucleases etc. can be added in an analogous manner.
- a nucleic acid amplification master mix which contains the amplification buffer, nucleotides, primers, polymerase and buffer salts can also be added during the elution.
- Step (f) of the process according to the invention comprises separating the eluate from the adsorption matrix. This separation can be carried out in the usual manner e.g. by sedimentation but preferably by magnetic separation.
- the analytes isolated by the process according to the invention can be subsequently processed further in a known manner e.g., in the case of nucleic acids by amplification and subsequent detection, or detection without previous amplification or sequencing.
- various analytes can be determined in aliquots of the eluate e.g. various viruses such as HIV, HCV and HBV.
- An important feature of the process according to the invention is that many or optionally even all steps can be carried out at essentially the same temperature i.e. within a temperature range of ⁇ 2.5° C.
- This temperature is preferably in the range of room temperature to 70° C., particular preferably from room temperature to 40° C., most preferably at room temperature i.e. ca. 18 to 32° C.
- at least the steps (c) of adsorption and (d) of washing are carried out at this temperature.
- Other steps, in particular the steps (a) of lysing or/and (e) of elution are particularly preferably also carried out at this temperature.
- the entire sample preparation can for example be carried out at a uniform temperature for the determination of HIV in blood samples.
- an additional after-treatment step at an elevated temperature can take place after step (f) of the process according to the invention which improves the amplification yields for certain analytes. It may be necessary for other analytes to carry out the pre-treatment or/and the elution at an elevated temperature.
- the elevated temperature is preferably in the range of more than 40° C. to 95° C. e.g. ca. 70° C.
- the process according to the invention is preferably carried out in an automated device. Examples of such devices are described in the following. It is also preferable that in the process according to the invention for sample preparation at least steps (a) to (e) are carried out in a single reaction vessel i.e. that there is no transfer into another reaction vessel. This considerably simplifies the process and also leads to a reduction of the risk of contamination.
- reagent kit which is especially suitable for carrying out the process described above comprising
- a reagent kit for isolating DNA comprising magnetic glass particles whose glass phase contains SiO 2 , B 2 O 3 and Na 2 O
- a reagent kit for isolating RNA comprising magnetic glass particles whose glass phase contains SiO 2 , B 2 O 3 , Al 2 O 3 , CaO and Ka 2 O.
- Another subject matter of the present invention is a device for isolating an analyte from a biological sample comprising:
- a first holding device for reaction vessels for sample preparation ( 3 ) which is equipped for an operating temperature of ⁇ 70° C., in particular ⁇ 40° C.
- reaction vessels 4 a , 4 b , 4 c
- a second holding device for reaction vessels 4 a , 4 b , 4 c
- optionally contains a cooling or/and heating means optionally contains a cooling or/and heating means
- the device according to the invention is preferably designed such that a single reaction vessel is used to carry out the 4 main steps of sample preparation i.e. lysis of a sample, adsorption of the released analyte e.g. a nucleic acid to a solid adsorption matrix e.g. magnetic glass particles, washing the adsorption matrix and eluting the analyte from the adsorption matrix.
- sample preparation i.e. lysis of a sample
- adsorption of the released analyte e.g. a nucleic acid
- a solid adsorption matrix e.g. magnetic glass particles
- the device is designed such that the first holding device for holding the reaction vessels for sample preparation is used at least for the adsorption of the analyte to the solid adsorption matrix and for washing the adsorption matrix.
- the first holding device is also used for the sample lysis or/and for the elution of the analyte from the adsorption matrix.
- the reaction vessels for the sample preparation have a volume of preferably at least 1 ml. e.g. 1-5 ml.
- the second holding device is designed for reaction vessels to store or/and further process the analyte e.g. PCR vessels which usually have a different shape than the reaction vessels used for the sample preparation.
- the reaction vessels for storing or/and additional processing have a volume of preferably up to 500 ⁇ l, e.g. 50-200 ⁇ l.
- the second holding device can contain vessels for reagents which are required to process the sample containing the analyte e.g. a PCR master mix.
- the device according to the invention can be designed such that one or several steps of the sample preparation or/and an after-treatment step can be carried out at an elevated temperature in the second holding device.
- the second holding device can be designed to hold reaction vessels for at least one treatment step which is selected from lysing the sample, eluting the sample from the adsorption matrix and an after-treatment step after elution.
- the first holding device preferably contains means for the magnetic separation.
- the first holding device contains means for mixing the reaction vessels in particular by rotating them around their longitudinal axis. Such means can optionally be provided for the second holding device.
- the robotic tool generally comprises automatic pipetting devices and optionally means for transporting reaction vessels e.g. between the first and second holding device.
- a cap opening and closing unit may be integrated.
- the sample preparation device ( 1 ) contains a holding device for reagents ( 2 ), a holding device for reaction vessels for sample preparation ( 3 ) with the functions mixing and magnetic separation which provides a temperature of preferably ⁇ 40° C. and particularly preferably room temperature.
- the device additionally contains a holding station for further reaction vessels ( 4 a ) e.g. for PCR vessels which has a temperature of 4° C. to room temperature.
- the device additionally contains automated devices for pipetting and handling reaction vessels ( 5 ) which enables movements in an X, Y and Z direction.
- the four main steps of sample preparation i.e.
- lysis, adsorption, washing and elution take place in a single reaction vessel in the first holding device. Eluates are stored and further reagents e.g. PCR master mix are added in the second holding device. For further processing e.g. for a subsequent PCR, the vessels are transferred to an appropriate device e.g. a thermocycler (not shown).
- an appropriate device e.g. a thermocycler (not shown).
- the device contains a second holding device ( 4 b ) which is designed to hold reaction vessels for further processing e.g. PCR vessels and is equipped to set a temperature of 4° C. (cooling the PCR master mix) to 95° C. to heat the eluate after elution from the adsorption matrix.
- a cap counter-heating is preferred to prevent condensation on the cap of the PCR vessels.
- the embodiment of the device according to the invention shown in FIG. 3 is provided with a second holding device for reaction vessels ( 4 c ) which is designed to hold PCR vessels or sample preparation vessels.
- a second holding device for reaction vessels 4 c
- cooling e.g. to 4° C. and heating e.g. to 95° C. is possible to heat the lysate or/and the eluate.
- a cap counter-heating is provided to prevent condensation on the cap of reaction vessels.
- the first holding device is designed to set a temperature in the range of ⁇ 70° C.
- the second holding device is—as shown in FIG. 3 —suitable for cooling and heating sample processing and sample preparation vessels.
- the devices according to the invention can be used especially in a process as described above.
- FIG. 1 shows a schematic representation of a first embodiment of the device according to the invention
- FIG. 2 shows a schematic representation of a second embodiment of the device according to the invention
- FIG. 3 shows a schematic representation of a third embodiment of the device according to the invention
- FIG. 4 shows the result of a chlamydia detection by PCR using manual and semiautomatic sample preparation
- FIG. 5 shows the result of a chlamydia detection by PCR using semiautomatic sample preparation and various temperature profiles during the sample preparation
- FIG. 6 shows the result of a HIV detection by PCR using manual sample preparation (standard protocol) and semiautomated sample preparation at room temperature.
- sols were prepared as follows:
- Alcoholates of the oxides were stirred together in the above molar ratios analogously to the procedure in examples 1 and 2 of WO96/41811 to form a homogeneous phase. However, a deviation was that no HCl was used.
- Alcoholates of the oxides were stirred together in the above molar ratios analogously to the procedure in examples 1 and 2 of WO96/41811 to form a homogeneous phase. However, a deviation was that no HCl was used.
- the sols were subsequently subjected to a spray drying process.
- the powder obtained by the spray drying was subjected to a separation of fines by sedimentation, a temperature treatment under a nitrogen atmosphere (60 l/h volume flow rate) at a heating rate of 1 K/min and kept for one hour at a compaction temperature in the range of 600 to 700° C. Subsequently the oven was cooled to 300° C. and flushed with oxygen for 1 h at this temperature. After cooling to room temperature the magnetic glass particles were removed and sieved through a 50 ⁇ m sieve to separate the coarse material.
- the magnetic glass particles obtained from sol 1 are particularly suitable for the isolation of DNA.
- the glass particles obtained from sol 2 are particularly suitable for the isolation of RNA.
- the following standard protocol is suitable for isolating nucleic acids from biological samples such as whole blood or cultured cells.
- the nucleic acids obtained in this manner can be used directly after the elution for an amplification by PCR, a restriction cleavage or a Southern blot.
- the reaction kit contains:
- binding buffer (4.7 mol/l guanidinium hydrochloride, 10 mmol/l urea, 10 mmol/l Tris HCl, 20% Triton®X-100, pH 5.7
- wash buffer (56% (v/v) ethanol, 20 mmol/l NaCl, 10 mmol/l Tris HCl, pH 7.5)
- the kit components are stable and can be stored at room temperature. After dissolving proteinase K in water, the solution should be aliquoted and stored at ⁇ 20° C. The frozen solution is stable for 12 months.
- 200 ⁇ l sample is added to a 2 ml reaction vessel and admixed with 200 ⁇ l binding buffer and 40 ⁇ l proteinase K solution. It is subsequently incubated for 10 min. The incubation is preferably carried out at room temperature. However, under certain circumstances the incubation temperature can also be increased to up to 70° C.
- reaction vessel is placed in a magnetic particle separator (Boehringer Mannheim, Cat. No. 1 641 794) and separated for about 1 min.
- a magnetic particle separator Boehringer Mannheim, Cat. No. 1 641 794
- Step 5 is repeated three times. After the last washing process the remaining wash buffer is completely removed.
- elution buffer which is optionally preheated to 70° C. is added. It is then mixed and incubated for 5 minutes at room temperature. The sample is placed in the MP separator and the supernatant is transferred into a clean reaction vessel.
- nucleic acids e.g. DNA obtained in this manner are stable and can be subsequently directly processed further or stored at 4° C.
- microtitre plates e.g. deep well microtitre plates (e.g. Ritter, J.J. Bioanalytic).
- 200 ⁇ l of a urine sample and 240 ⁇ l binding buffer/proteinase K solution (5:1) are pipetted into a 2 ml reaction vessel, subjected to vortex mixing and incubated for 10 min at 70° C. Then the sample is cooled for 5 min to room temperature.
- the MGPs are concentrated by transferring the sample to a magnetic separator. After one minute the supernatant is completely removed by pipette.
- 0.5 ml wash buffer is added to the MGPs by pipette.
- the sample is subjected to vortex mixing and then transferred to the magnetic separator.
- the supernatant is removed by pipette after 1 min.
- the washing procedure is repeated for a further two times.
- elution buffer 200 ml elution buffer is added to the MGP.
- the sample is incubated for 10 min at 70° C. in a thermomixer at 1400 rpm. Condensed water is collected by briefly centrifuging. The sample is transferred to the magnetic separator and after 1 min 180 ⁇ l eluate is removed. The eluate is pipetted into a new reaction vessel and stored at 4° C. (for a storage period of ⁇ 24 h) or at ⁇ 20° C. (for a longer storage period).
- FIG. 4 shows a comparison of the determination of chlamydia. (sample: 100 elementary antibodies per 100 ml urine; six-fold determination) between the manual standard protocol (vortex) and the semiautomated process (MTM). It can be seen that the sensitivity is not impaired by the automation.
- the sample preparation is carried out as described in section 3.2. However, the lysis and elution are carried out at room temperature.
- the sample preparation is carried out as described in section 3.3. After elution an incubation is carried out for 10 min at 70° C.
- FIG. 5 shows a comparison of the chlamydia determination (samples: SWE1, O chlamydia elementary antibodies (EAB) per ml urine, SWE2: 10 EAB, SWE3: 100 EAB and SWE4:1000 EAB each per ml urine) between the sample preparation protocols described in sections 3.2, 3.3 and 3.4.
- the standard protocol is more sensitive compared to a sample preparation at room temperature (RT protocol MTM) for the determination of chlamydia.
- RT protocol MTM sample preparation at room temperature
- the results of the sample preparation at room temperature and subsequent aftertreatment of the eluate (RT protocol MTM with aftertreatment) show that this effect can be largely compensated. It is therefore surprising that a temperature step is not necessary during the sample preparation per se.
- Frozen plasma is thawed for 5 min at 37° C. and cooled on ice for further processing.
- a proteinase K solution (25 mg/ml) is pipetted into a 1.5 ml Sarstedt reaction vessel. 250 ⁇ l sample is added to this and mixed in a vortex mixer. Then 300 ⁇ l lysis buffer is added and it is again vortex mixed.
- wash buffer 750 ⁇ l wash buffer is added to the MGPs.
- the MGPs are resuspended and separated as described previously.
- the wash procedure is repeated four times and the washing buffer is carefully removed at the end.
- the sample preparation is carried out as described in section 4.1 except that the mixing and heating was carried out on a mixing and heating module.
- the sample preparation is essentially carried out as described in section 4.2 except that all steps are carried out at room temperature.
- the incubation period for lysis, adsorption and elution is in each case 15 min.
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US10/426,641 US20030199078A1 (en) | 1997-10-01 | 2003-04-30 | Method, kit and apparatus for the isolation of nucleic acids |
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DE19743518A DE19743518A1 (de) | 1997-10-01 | 1997-10-01 | Automatisierbare universell anwendbare Probenvorbereitungsmethode |
DE19743518 | 1997-10-01 | ||
PCT/EP1998/006196 WO1999016781A2 (fr) | 1997-10-01 | 1998-09-29 | Procede de preparation d'echantillons automatisable, a usage universel |
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ES2398713T3 (es) | 2013-03-21 |
JP2001518284A (ja) | 2001-10-16 |
WO1999016781A2 (fr) | 1999-04-08 |
ES2290996T3 (es) | 2008-02-16 |
EP1783135A1 (fr) | 2007-05-09 |
KR20010024366A (ko) | 2001-03-26 |
DE59814056D1 (de) | 2007-08-23 |
JP4048022B2 (ja) | 2008-02-13 |
DE19743518A1 (de) | 1999-04-15 |
ATE366739T1 (de) | 2007-08-15 |
CA2305171C (fr) | 2008-06-17 |
EP1783135B1 (fr) | 2012-11-07 |
DK1019430T3 (da) | 2007-11-12 |
JP4659013B2 (ja) | 2011-03-30 |
JP2007175060A (ja) | 2007-07-12 |
JP2008134239A (ja) | 2008-06-12 |
PT1019430E (pt) | 2007-10-16 |
US20030199078A1 (en) | 2003-10-23 |
EP1019430A2 (fr) | 2000-07-19 |
WO1999016781A3 (fr) | 1999-09-16 |
AU1028299A (en) | 1999-04-23 |
CA2305171A1 (fr) | 1999-04-08 |
IL135299A (en) | 2010-11-30 |
IL135299A0 (en) | 2001-05-20 |
EP1019430B1 (fr) | 2007-07-11 |
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